【作者】 武亮；

【导师】 邓昌彦；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 堆肥是处理畜禽场废弃物的主要手段之一,这种处理方法具有投资小,效益高,可以使大量有机固体废弃物重复资源化利用等优点,但是同时也存在发酵周期长、堆肥产品质量不高等缺陷。为了提高堆肥产品的质量、缩短堆肥周期、提高堆肥效率,本试验主要从堆肥调理剂的选择、堆肥微生物菌种的筛选、筛选菌株的功能及对堆肥中微生物群落的影响等方面开展了研究,试验结果总结如下：1.不同调理剂对猪粪好氧堆肥过程的影响分别以锯末、米糠和木屑为调理剂,调节堆肥原料的碳氮比为25：1,含水率为60%,通风方式采用人工翻堆的方式进行猪粪好氧堆肥。从各调理剂堆肥的温度、含水率、pH值、有机碳含量、发芽指数、全氮含量及碳氮比等方面进行了比较。3个堆体都经历了快速升温期、持续高温期和缓慢降温期3个时期,均可以实现好氧堆肥的腐熟,并使堆肥产品达到国家粪便无害化卫生标准。不同的调理剂对猪粪好氧堆肥堆体的温度变化影响较大,其中,以锯末为调理剂的堆体升温最快,米糠次之,木屑升温最慢。以米糠为调理剂的堆体的高温期最长,锯末次之,木屑的高温期最短。3个堆体含水率的变化趋势相同,都随着堆肥的进程而不断降低,都从开始的60%左右降低到33%左右。使用3种不同调理剂进行猪粪好氧堆肥的最终产品中氮元素的相对含量差异显著(P<0.05),以锯末和米糠为调理剂的堆肥产品的全氮含量较高,为2.12%和2.10%,以木屑为调理剂的堆肥产品的的全氮含量较低,为1.90%。2.堆肥中产葡聚糖内切酶微生物的筛选、分离与纯化通过以CMC-Na为唯一碳源的筛选培养基进行初筛得到17株不同的微生物,经滤纸培养基进行复筛后得到一株能够产CMC酶的菌株。经形态学鉴定发现,该菌株为产芽孢的革兰氏阳性菌,并通过细菌16S rDNA的通用引物对该菌株的16SrDNA扩增测序,与NCBI数据库中的序列比对后发现,该菌株的16SrDNA序列与枯草芽孢杆菌的16SrDNA序列相似度达到100%,因此该菌株被鉴定为枯草芽孢杆菌,命名为Subtilis bacillus dcy-1,并保藏在中国典型培养物保藏中心(CCTCC),保藏编号为：CCTCC M208122。分离菌株的最佳液体产酶培养基为：麸皮1.5%,豆粕1.5%,NaCl 0.5%, CaCl2 0.05%, MgSO4 0.05%,酵母提取物0.1%；产酶条件为：发酵温度为30℃,培养基初始pH值为7.0,培养时间为40h时CMC酶活达到最大。菌株所产的CMC酶的最佳作用温度为55℃,最佳pH值为7.5,Na+、Ca2+、Ni2+对CMC酶酶促反应有一定的促进作用,Cu2+和Mn2+对CMC酶酶促反应有显著的抑制作用。CMC酶在低于60℃时热稳定性较好,在pH值为5-8时比较稳定。3.分离并鉴定了枯草芽孢杆菌Subtilis Bacillus-dcyl的Egl基因通过设计引物获得了完整的CDS序列,GenBank数据库登陆号为,HM036060,通过生物信息学分析软件对基因的结构和性质进行了预测。以枯草芽孢杆菌为出发菌株,构建了枯草芽孢杆菌Egl基因重组原核表达载体pGEX-KG,优化后的最佳条件为IPTG诱导3.5h,其浓度为0.6mmol/L,主要以可溶的形式存在,通过对表达产物活性的测定发现,葡聚糖内切酶活性可达121.23U/ml。4.堆肥中微生物总DNA提取方法的改进研究针对堆肥中微生物抗性比较强的特点,设计了通过蛋白酶和蜗牛酶2次消化的堆肥样品的DNA提取方法。分别从对微生物细胞的裂解效率、DNA产量、DNA回收效率及PCR可扩增性等方面对3种不同的DNA提取方法进行了研究比较,结果表明,3种方法都能够从堆肥样品中成功的提取出微生物的DNA,但是,双酶裂解法比其他2种方法效率更高,更加经济实用。本试验中所提出和改进DNA提取方法可以有效的应用于堆肥样品中,由于堆肥样品中的杂质和干扰物要比土壤、污水等环境样品中更加复杂一些,所以,这种方法也可以应用于其他环境样品微生物的提取与纯化。5.添加外源微生物对堆肥细菌群落的影响堆体接种外源微生物后能够明显的提高堆体的温度升高速度和堆体的最高温度,延长了高温期的时间。通过RFLP技术快速、准确地研究了猪粪好氧堆肥过程中不同时期的细菌群落的变化。比较了接种外源微生物的堆体和自然堆肥堆体中细菌种类和数量的差异,结果表明接种外源微生物可以改变堆肥中微生物群落的变化,所接种的微生物能够适应堆肥的环境,并成为优势菌群。通过与NCBI数据库中比较,试验中共发现35条序列与已知微生物序列相似性很低,这些序列有可能是堆肥中尚未发现的新物种,它们在堆肥中的作用有待进行进一步研究。更多还原

【Abstract】 Compost is a primary treatment for the waste of livestock. This method is efficiency and resourceful to deal with organic solid wste, but it also have some disadvatages, for exampl, long time for fermentation and low qulity. In order to improve the quality of compost products, shorten the composting time and improve the composting efficiency, this study reserch in the choice of compost conditioner, screening for compost microbial strains and the impact of the strains for microbial community in compost mainly.The results are summarized as follows:1. Different Conditions on aerobic composting of pig manureRespectively, sawdust, rice bran and wood chipper were used as the conditioner for swin composting. The initial carbon and nitrogen ratio was 25:1. the moisture content of the mixture was 60% and the ventilation method was turning pig manure composting. We compared the difference in temperature, moisture, pH, organic carbon, germination.index, total nitrogen and nitrogen ratio. Three piles experienced a rapid warming stage, high temperature stage and slow cooling stage. All cmpost piles meet the national standard for fecal harmless. Different Conditions influenced the temperature of composting pile. With sawdust as bulking agents in the pile heat up the fastest, followed by rice bran. Wood chips was the slowest. With rice bran as the conditioning agent experenced the longest high-temperature stage, followed by sawdust, wood chips is the shortest one. The trend of moisture content of three piles reduced with the composting process. All piles reduced to about 30% from 60%. Use different conditioning agents in the pig manure composting lead to a significantly different (P<0.05) content of nitrogen. Total nitrogen content of compost with sawdust, rice bran and wood chipers were 2.12%,2.10% and 1.90% respectely.2. Screening, isolation and purification for endo-glucanase enzymes produced strains in compostAfter growing in screening medium in which CMC-Na was the only carbon source we get 17 different microorganisms. All this microorganisms were screened again by filter paper medium we finally obtained an CMCase produced strain. After observed by the culture, Gram staining, spores staining and compareing 16S rRNA with NCBI database the screening strain was identified as Bacillus subtilis and named Subtilis bacillus dcy-1, and preserved in the China Center for Type CultureCollection (CCTCC), preservation code:CCTCC M 208122. The best liquid medium for enzyme production was:wheat bran 1.5%, soybean meal 1.5%, NaCl 0.5%, CaCl2 0.05%, MgSO4 0.05%, yeast extract 0.1%. The best fermentation conditions were:fermentation temperature is 30℃, initial pH 7.0 and the fermentation time were 40h. The best reaction temperature for enzym activity was 55℃, the best pH value was 7.5, Na-, Ca2+, Ni2+ would improve enzyme activity and Cu2+ and Mn2+ would inhibit enzyme activity. Endo-glucanse was good heat stability in less than 60℃and pH 5-8.3. Isolated and identified Egl gene from subtilis bacillus which separated from pig manure composting, complete CDS sequence obtained, GenBank database accession number is, predicted structure and properties of the gene by bioinformatics analysis software were.Constructed recombinant prokaryotic expression vector pGEX-KG of the Egl gene of Bacillus subtilis dcy-1 was constucted. The optimum conditions for expression were:the concentration of IPTG was 0.6mmol/L and the induction time was 3.5h. The activitys of the expressed products were 121.23U/ml.4. Improvement for DNA Extraction method of microorganism in compostBased on the strong resistance of microorganism in compost, designed that samples of compost were digested by protease and helicase. An effective cell lysis method for extraction of bacterial genomic DNA from compost was developed in this study. Enzymatic disruption method, physical-chemical combination method, and commercial kit method were used to extract DNA from compost samples and were compared by analyzing DNA yield and efficient cell lysis. The results showed that all the three methods can be used to extract high-quality DNA from compost, but the enzymatic method had better cell lysis efficiency and DNA yields than others without the use of special equipment and expensive spending. Comparison of different methods for lysing gram-positive bacteria Bacillus subtilis indicated that the enzymatic cell lysis is superior for destroying the gram-positive cell wall. Spin-bind DNA column was used for DNA purification, and the purity of the purified sample was checked by polymerase chain reaction to amplify a region of the 16S rRNA. Results indicated that the part of 16S rRNA was amplified from all the purified DNA samples, and all the amplification products could be digested by the restriction enzyme Hhal.5. Adding exogenous microbial communities of bacteria in compostThe temperature of-compost can significantly increase when inoculated the microorganisms. Reserched the diversity of bacterial communities in compost by RFLP technique. Compared the difference on bacerial species and qualtity between inoculated and without inoculated composte piles. Results showed that the microorganisms communities can be changed when inoculated exogenous bacterial to cmopost. The exogenous bacterial can grow rapidly in compost and improve to be predominated communities.35 sequences were found, have no similarity with synxenic in th genbank data, this new sequeces may be repreasentation of new spices and the role they played in compst would be worth for further research.更多还原